U.S. patent application number 13/518787 was filed with the patent office on 2013-02-07 for obtainment of bioactive products from cocoa having inhibitory activity against the pep enzyme and antioxidant and/or antineurodegenerative activity.
This patent application is currently assigned to NATRACEUTICAL INDUSTRIAL S.L.U.. The applicant listed for this patent is Esther Bataller Leiva, Salvador Genoves Martinez, Nuria Gonzalez Martinez, Aida Ibannez Lopez, Silvia Llopis Pla, Patricia Martorell Guerola, Honorato Monzo Oltra, Begona Muguerza Marquinez, Daniel Ramon Vidal. Invention is credited to Esther Bataller Leiva, Salvador Genoves Martinez, Nuria Gonzalez Martinez, Aida Ibannez Lopez, Silvia Llopis Pla, Patricia Martorell Guerola, Honorato Monzo Oltra, Begona Muguerza Marquinez, Daniel Ramon Vidal.
Application Number | 20130035291 13/518787 |
Document ID | / |
Family ID | 44194987 |
Filed Date | 2013-02-07 |
United States Patent
Application |
20130035291 |
Kind Code |
A1 |
Bataller Leiva; Esther ; et
al. |
February 7, 2013 |
OBTAINMENT OF BIOACTIVE PRODUCTS FROM COCOA HAVING INHIBITORY
ACTIVITY AGAINST THE PEP ENZYME AND ANTIOXIDANT AND/OR
ANTINEURODEGENERATIVE ACTIVITY
Abstract
The present invention relates to the production of bioactive
products from raw plant matter, namely cocoa extracts. The said
products have one or more biopeptides with prolyl endopeptidase
(PEP) enzyme inhibitory activity in vitro and/or antioxidant and/or
antineurodegenerative capacity in vivo and can be used in dietetics
and in the food and pharmaceutical industries.
Inventors: |
Bataller Leiva; Esther;
(Valterna-Paterna (Valencia), ES) ; Genoves Martinez;
Salvador; (Aldaia (Valencia), ES) ; Martorell
Guerola; Patricia; (Picassent (Valencia), ES) ; Ramon
Vidal; Daniel; (L'Eliana (Valencia), ES) ; Ibannez
Lopez; Aida; (Benisano (Valenica), ES) ; Llopis Pla;
Silvia; (Guadassequies (Valencia), ES) ; Gonzalez
Martinez; Nuria; (Alacuas (Valencia), ES) ; Monzo
Oltra; Honorato; (La Canyada-Paterna (Valencia), ES)
; Muguerza Marquinez; Begona; (Valencia, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bataller Leiva; Esther
Genoves Martinez; Salvador
Martorell Guerola; Patricia
Ramon Vidal; Daniel
Ibannez Lopez; Aida
Llopis Pla; Silvia
Gonzalez Martinez; Nuria
Monzo Oltra; Honorato
Muguerza Marquinez; Begona |
Valterna-Paterna (Valencia)
Aldaia (Valencia)
Picassent (Valencia)
L'Eliana (Valencia)
Benisano (Valenica)
Guadassequies (Valencia)
Alacuas (Valencia)
La Canyada-Paterna (Valencia)
Valencia |
|
ES
ES
ES
ES
ES
ES
ES
ES
ES |
|
|
Assignee: |
NATRACEUTICAL INDUSTRIAL
S.L.U.
Quart de Poblet (Valencia)
ES
BIOPOLIS, S.L.
Paterna (Valencia)
ES
|
Family ID: |
44194987 |
Appl. No.: |
13/518787 |
Filed: |
December 23, 2009 |
PCT Filed: |
December 23, 2009 |
PCT NO: |
PCT/ES2009/000597 |
371 Date: |
October 24, 2012 |
Current U.S.
Class: |
514/15.7 ;
435/68.1; 514/21.4; 514/21.5; 514/21.6; 514/21.7; 514/21.8;
530/326; 530/327; 530/328; 530/329; 530/330 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 39/06 20180101; A23G 1/44 20130101; A23L 33/105 20160801; A61P
9/12 20180101 |
Class at
Publication: |
514/15.7 ;
435/68.1; 514/21.8; 514/21.7; 514/21.6; 514/21.5; 514/21.4;
530/330; 530/329; 530/328; 530/327; 530/326 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61P 9/12 20060101 A61P009/12; C07K 7/08 20060101
C07K007/08; A61P 39/06 20060101 A61P039/06; C07K 7/06 20060101
C07K007/06; C12P 21/06 20060101 C12P021/06; A61K 38/08 20060101
A61K038/08 |
Claims
1-11. (canceled)
12. Bioactive product with PEP enzyme inhibitory activity in vitro
and antioxidant and/or anti-neurodegenerative capacity in vivo,
characterised in that it comprises one or more peptides formed by a
chain of 5 to 20 amino acids selected from the group consisting of:
SEQ. ID. No. 1; SEQ. ID. No. 2; SEQ. ID. No. 3; SEQ. ID. No. 4;
SEQ. ID. No. 5; SEQ. ID. No. 6; SEQ. ID. No. 7; SEQ. ID. No. 8;
SEQ. ID. No. 10; SEQ. ID. No. 12; SEQ. ID. No. 13; SEQ. ID. No. 14;
SEQ. ID. No. 15; SEQ. ID. No. 16; SEQ. ID. No. 17; SEQ. ID. No. 19;
SEQ. ID. No. 20; SEQ. ID. No. 22; SEQ. ID. No. 23; SEQ. ID. No. 24;
SEQ. ID. No. 25; SEQ. ID. No. 26; SEQ. ID. No. 27 and SEQ. ID. No.
28; with the exception of bioactive products comprising one or more
peptides selected from the group consisting of: SDNEWAWMF (SEQ.ID.
No. 29); LSDNEWAWMF (SEQ. ID. No. 30); SDNEWAWMFK (SEQ. ID. No.
31); LSDNEWAWMFK (SEQ. ID. No. 32); RRSDLDNGTPVIF (SEQ. ID. No.
33); DNYDNSAGKWWVT (SEQ. ID. No. 34); TSTVWRLDNYDNSA (SEQ. ID. No.
35) and DNYDNSAGKWWVTTD (SEQ. ID. No. 36).
13. The bioactive product according to claim 12 obtained from cocoa
extracts.
14. The bioactive product obtained from cocoa extracts according to
claim 13, characterised in that it is obtained by enzymatic
hydrolysis from a cocoa product with husk, referred to as bark.
15. The bioactive product according to claim 13, characterised in
that prior to enzymatic hydrolysis the bark has been defatted by
means of physical pressing, until having between 5% and 15% fat,
preferably 10%.
16. The bioactive product according to claim 13 characterised in
that the defatted product is dissolved in water (5% to 20%) and is
subjected to a step of enzymatic hydrolysis, in which one or more
enzyme/enzymes is/are added to the aqueous solution obtained and
the temperature is maintained at between 40.degree. C. and
60.degree. C. for a time period lasting from 1 to 24 hours, at a
suitable pH.
17. The bioactive product according to claim 13 characterised in
that enzymatic hydrolysis is performed by one or more
enzyme/enzymes belonging to the Termamyl, Alcalase, Neutrase,
Ultraflo or Flavourzyme group.
18. The bioactive product according to claim 13 characterised in
that the bioactive peptides are obtained by centrifugation, from
the supernatant of the hydrolyzed extract, once hydrolysis has
finished.
19. The use of the bioactive product according to claim 12, for the
manufacture of food or a functional ingredient with
antihypertensive and/or anti-degenerative and/or antioxidant
activities.
20. Functional food with PEP enzyme inhibitory activity in vitro
and antioxidant and/or anti-neurodegenerative capacity in vivo,
characterised in that it comprises the bioactive product according
to claim 12.
21. Use of the bioactive product according to claim 12 for the
manufacture of a medicine to treat neurodegenerative
conditions.
22. A pharmaceutical composition that comprises the bioactive
product according to claim 12, characterised in that said
composition has antihypertensive and/or antidegenerative and/or
antioxidant activity.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to the production of bioactive
products from raw plant matter, namely cocoa extracts. The said
products have one or more biopeptides with prolyl endopeptidase
(PEP) enzyme inhibitory activity in vitro and/or antioxidant and/or
antineurodegenerative capacity in vivo and can be used in dietetics
and in the food and pharmaceutical industries.
BACKGROUND OF THE INVENTION
[0002] This application can be considered a continuation
application of patent application PCT/ES2008/000540, by the same
authors of the present invention, wherein bioactive products
obtained by enzymatic hydrolysis are described, comprising one or
more of a series of eight peptides that have been isolated and
characterised therein by their sequences. The sequences of these
eight peptides are the following:
TABLE-US-00001 (SEQ. ID. No. 29) SDNEWAWMF; (SEQ. ID. No. 30)
LSDNEWAWMF; (SEQ. ID. No. 31) SDNEWAWMFK; (SEQ. ID. No. 32)
LSDNEWAWMFK; (SEQ. ID. No. 33) RRSDLDNGTPVIF; (SEQ. ID. No. 34)
DNYDNSAGKWWVT; (SEQ. ID. No. 35) TSTVWRLDNYDNSA and (SEQ. ID. No.
36) DNYDNSAGKWWVTTD.
[0003] The proteins in foods are precursors of a large number of
peptides with special biological activity, having useful properties
affecting different bodily processes. Due to the great interest in
developing natural products that provide some sort of "extra"
beneficial health effect for persons consuming them, there is a
good opportunity to use various raw plant matter sources to develop
new products and functional ingredients. The possibility of
generating these peptides is a state-of-the-art research field in
the nutraceutical industry as it enables the generation of new
functional food applications, giving rise to the added value of
food components and byproducts, improving the nutritive properties
of conventional foods and developing new dietary supplements or
even new medicinal products.
[0004] Some byproducts of the agro-food industry have a high
content of proteins and bioactive peptides, conferring added value
to the same. There are different mechanisms to enhance increased
peptide content by considerably enriching a matrix and conferring
to it specific characteristics, such as for example,
antihypertensive, antihyperglycemic, antineurodegenerative,
anticariogenic, and even antihyperlipidemic characteristics.
[0005] Proteolytic enzymes can be used to hydrolyze proteins at
specific points, capable of generating a broad range of peptides in
the hydrolyzed products obtained with multiple physiological
effects.
[0006] References to the state of the art can be found in the
following documents, among others: the patent application
WO91/19800 A1, which relates to proteins and nucleic acids derived
from cocoa. Specifically, claims relate to fragments of the protein
albumin (21 kDa), from the cacao seed, as cocoa aroma precursors.
Said patent application assigns to the peptide of 205 amino acids,
or fragments of the same, the functionality of being precursors of
cocoa flavour.
[0007] European patent EP1298210 A1 describes peptides derived from
albumin and vicilin proteins derived from the cocoa bean as
chocolate flavour precursors. The authors of the aforementioned
document attribute to the peptides, of between 2 and 30 amino
acids, preferably between 2 and 5 amino acids, the functionality of
being precursors of chocolate flavour. Likewise, said authors also
claim the use of these peptides for the manufacture of ice-cream,
beverages, dairy products, cosmetics, animal feed and
pharmaceuticals.
[0008] Patent application WO96/38472 A1 claims the peptides that
are cocoa flavour precursors to be the peptides with the amino acid
sequence Lys-Ala-Pro-Leu-Ser-Pro-Gly-Asp-Val-Phe-Val and fragments
from 2 to 11 amino acids contained in the said sequence. The
claimed sequence and fragments thereof are attributed the
functionality of being cocoa flavour precursors.
[0009] Patent application WO02/42327 A2 relates to the process of
isolation, purification and identification of 2S albumin protein,
as cocoa flavour precursor protein. Specifically, said application
describes the use of the said polypeptide or fragments thereof for
the production of cocoa/chocolate flavour.
[0010] Patent document JP2008019228 describes an amino acid
composition derived from cocoa extracts subjected to prior
extraction of polyphenols with angiotensin-1 converting enzyme
(ACE) inhibitory properties, as well as the foods and foodstuffs
containing the said composition. The said document does not specify
the peptide/amino acid sequence comprised by the said extract nor
does it mention the isolation and/or purification process
employed.
[0011] Prolyl-endopeptidase (PEP) enzyme activity is associated
with loss of memory and learning processes because it degrades the
proline-rich neuropeptides, such as vasopressin and substance P,
involved in these processes. Some studies also indicate that this
enzyme could be linked to Alzheimer's disease. To date, several
authors such as Kim et al., ("Prolyl Endopeptidase Inhibitors from
Green Tea", Arch Pharm Res, 24 (4):292-296 2001) and Tezuka et al.
("Screening of crude drug extracts for prolylendopeptidase
inhibitory activity", Phytomedicine, 6(3):197-203 1999), assert
that the polyphenol extracts of certain plants cause prolyl
endopeptidase inhibition. Patent document US 2007/0116779 also
describes the use of cocoa beans as a polyphenol source, as one of
the elements of pharmaceutical compositions and as an inhibitor of
certain enzymes involved in neurodegenerative diseases, such as
Alzheimer's or Parkinson's.
[0012] Other authors, such as Maruyama et al., ("Prolyl
Endopeptidase Inhibitory Activity of Peptides in the Repeated
Sequence of various Proline-Rich proteins", Journal of Fermentation
and Bioengineering, 74:145-148 1992) and Asano et al., ("Inhibition
of prolyl endopeptidase by synthetic peptide fragments of human
beta-casein"; Agric.Biol.Chem, 55(3):825-828 1991) state that some
peptide fragments inhibit PEP activity; however, to date there are
no references to cocoa peptides as inhibitory metabolites of the
said enzyme.
[0013] Therefore, due to the increasing demand for new ingredients
and compounds with this kind of inhibitory activity, given the
healthy characteristics of cocoa, the possibility was considered of
finding bioactive peptides from cocoa extracts that exert PEP
inhibitory activity. Eight peptides were isolated that not only
have PEP inhibitory activity in vitro, but said bioactive peptides
also show antioxidant and/or antineurodegenerative capacity in vivo
according to assays with the nematode model C. elegans, which gave
rise to the patent application PCT/ES/000540.
[0014] Ongoing research has surprisingly found new bioactive
products comprising 28 new peptides.
OBJECT OF THE INVENTION
[0015] The present invention relates to the production of bioactive
products from cocoa extracts rich in new bioactive peptides with
PEP enzyme inhibitory activity in vitro and/or antioxidant and/or
antineurodegenerative capacity in vivo.
[0016] Specifically, the invention relates to obtaining purified
peptide fractions from cocoa that have PEP enzyme inhibitory
activity and/or antioxidant and/or antineurodegenerative capacity
for potential use as ingredients to be incorporated in functional
foods.
[0017] The bioactive peptides can be produced by hydrolysis of one
or more proteins or peptides present in the cocoa extracts
obtained. For this purpose, enzymes and hydrolysis conditions are
set to obtain the desired biopeptides.
[0018] Different methodologies may be used to study these
inhibitory activities. In vitro tests are based on the
determination of PEP enzyme enzymatic activity in the presence of
the inhibitor in question, i.e., in the present invention this
relates to the peptide-rich cocoa fractions.
[0019] The bioactive peptides or the hydrolysates containing them
can be incorporated into functional foods. Once concentrated, the
fractions can be used in the food industry and in dietary products
as well as in the pharmaceutical industry for medicinal product
manufacture. The said bioactive products containing a suitable
amount of the said peptides can be used in the treatment and
prevention of diseases, such as blood pressure control, namely
hypertension, and also degenerative-type diseases such as
Alzheimer's, Parkinson's, etc. They can also be used as
antioxidants. Thus, the invention finds new applications for cocoa,
contributing to its positive reassessment from the health
viewpoint.
[0020] The 28 peptides, object of this invention, that the
bioactive products may contain which, on having been sequenced and
identified, can be obtained from any raw material source or
substrate that contains them. In addition, the new bioactive
peptides identified in the hydrolysates can be obtained by chemical
and/or enzymatic peptide synthesis or by recombinant methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Hydrophobic interaction chromatograph (HIC). On
observing the curve at 214 nm, it is concluded that all the protein
is contained in fractions 3 and 4.
[0022] FIG. 2. Reversed-phase chromatograph of HIC (hydrophobic
interaction chromatography) fraction 3. On observing the curve at
214 nm, it can be concluded that all the protein is contained in
fractions 7 and 11.
[0023] FIG. 3. Reversed-phase chromatograph of HIC fraction 4. On
observing the curve at 214 nm, it can be concluded that all the
protein is contained in fractions 7 and 11.
[0024] FIG. 4. Assay of the protective capacity against the
accumulation of amyloid-beta peptide in C. elegans CL4176 with
fractions F8 (31.3 .mu.g protein/ml); F9 (18.1 .mu.g protein/ml)
and F10 (9.9 .mu.g protein/ml). Positive controls were ZPP (0.1 mM)
and vitamin C (0.1 mg/ml). From this figure it can be concluded
that fractions F8, F9 and F10 provide protection against the
accumulation of B-amyloid petide, with F9 and F10 being the
fractions conferring the greatest protection.
[0025] FIG. 5. Effect of peptide extracts F8 (31.3 .mu.g
protein/ml); F9 (18.1 .mu.g protein/ml) and F10 (9.9 .mu.g
protein/ml) on survival in C. elegans CL2070 strain. Positive
controls were ZPP (0.1 mM) and vitamin C (0.1 mg/ml). The bars
indicate standard deviations. The figure shows how when subjected
to oxidative stress conditions, there was an increase in survival
rates of nematodes treated with peptide extracts from fractions F8,
F9 and F10.
[0026] FIG. 6. Effect of peptide extracts F8 (31.3 .mu.g
protein/ml), F9 (18.1 .mu.g protein/ml) and F10 (9.9 .mu.g
protein/ml) on the survival of the nematodes of the C.elegans
CL2070 strain. ZPP (0.1 mM) and vitamin C (0.1 mg/ml) were used as
positive controls. This figure shows the increased survival curve
corresponding to nematodes treated with peptide extracts from
fractions F8, F9 and F10.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention relates to a method to obtain
bioactive products, specifically from fractions of cocoa rich in
new peptides with PEP inhibitory activity in vitro and/or
antioxidant and/or antineurodegenerative capacity in vivo.
Specifically, the invention is based on obtaining different peptide
fractions by enzymatic hydrolysis of a cocoa byproduct (bark) to
obtain bioactive peptides, with the purpose of their potential use
as ingredients for incorporation into functional foods
[0028] Another aspect of the invention relates to purification of
the peptides from the cocoa extract obtained. Different
purification strategies are used depending on the peptides present
in the extract, for example, concentration by ion-exchange or
hydrophobic interaction, and separation, for example by RPC
(reverse phase chromatography) or gel filtration
chromatography.
[0029] The present invention relates to 28 new bioactive peptides
from cocoa extracts. The said bioactive peptides are identified
with the amino acid sequences denoted: SEQ. ID. No. 1; SEQ. ID. No.
2; SEQ. ID. No. 3; SEQ. ID. No. 4; SEQ. ID. No. 5; SEQ. ID. No. 6;
SEQ. ID. No. 7; SEQ. ID. No. 8; SEQ. ID. No. 9; SEQ. ID. No. 10;
SEQ. ID. No. 11; SEQ. ID. No. 12; SEQ. ID. No. 13; SEQ. ID. No. 14;
SEQ. ID. No. 15; SEQ. ID. No. 16; SEQ. ID. No. 17; SEQ. ID. No. 18;
SEQ. ID. No. 19; SEQ. ID. No. 20; SEQ. ID. No. 21; SEQ. ID. No. 22;
SEQ. ID. No. 23; SEQ. ID. No. 24; SEQ. ID. No. 25; SEQ. ID. No. 26;
SEQ. ID. No. 27 and SEQ. ID. No. 28, which have PEP enzyme
inhibitory activity in vitro enzyme and/or antioxidant and/or
antineurodegenerative capacity in vivo.
[0030] Furthermore, the invention relates to the use of the said
bioactive peptides as functional ingredients for different foods,
considering foods to be any composition intended for consumption;
be it liquid or solid.
[0031] The raw material source of the present invention may be any
appropriate substrate comprising one or more of the bioactive
peptides described herein. In a particular embodiment, the said raw
material is a plant, namely the raw material source is cocoa.
Theobroma cacao is the scientific name of the cacao tree or cocoa
tree. The term cocoa comes from Mayan (Ka'kaw) and Theobroma is
Greek for "food of the gods". More specifically a cocoa byproduct
called bark is used, which is cocoa with the husk partially
defatted by means of physical pressing, with a fat proportion that
can range between 5% and 15%, preferably 10%, expressed in weight
percentage.
[0032] The said raw material source is dissolved in water at a rate
that can vary between 5% and 20%; preferably 10%. Once the bark is
dissolved in water it is treated at different temperatures; for
example between 40.degree. C. and 60.degree. C., preferably
50.degree. C. for different time periods; for example between 1 and
24 hours; preferably 1, 6, 18 or 24 hours with one or more
hydrolytic enzymes at different concentrations. Any enzyme capable
of providing the peptides of interest can be used. Specifically,
the enzymes used are equally enzymes with cellulose activity, as is
the case of the enzyme Termamyl, and enzymes with protease
activity, such as the enzymes Alcalase, Neutrase, Ultraflo or
Flavourzyme. A single enzyme or combinations of two or more enzymes
can be used, provided that they produce one or more biopeptides
with the desired characteristics. The enzyme concentrations used
range between 0.10 and 10 .mu.l/L, and between 0.5 and 2 .mu.l of
enzyme per g of raw material. The hydrolysis conditions are: pH,
temperature, pressure, concentration of enzyme(s), reaction time,
etc. are optimized depending on the enzyme or enzymes used.
[0033] To obtain the extract, once the hydrolysis reaction has
ended, the enzyme/enzymes is/are inactivated. Centrifugation at
4000 rpm is performed for 15 minutes and the supernatant containing
the bioactive peptides, object of the present invention, is
collected and then separated into fractions and subfractions for
isolation and purification.
[0034] Therefore, another aspect of the invention relates to the
purification and identification of the bioactive peptides obtained
from the cocoa extracts. Given that the peptides present in the
extract are of a different amino acid length and considering that
the bioactive peptides of interest have between 5 and 20 amino
acids, different purification strategies can be carried out both
for removing unwanted peptides and for isolating and concentrating
the peptides object of the invention. For example, fractions of a
different molecular weight can be obtained from the hydrolyzed
products by means of ultrafiltration, with subsequent active
subfraction isolation by means of ion exchange or hydrophobic
interaction or high performance reverse phase or gel filtration
chromatography.
[0035] The advantages of the present invention are not only due to
the new peptides previously referred to as SEQ. ID. No. 1; SEQ. ID.
No. 2; SEQ. ID. No. 3; SEQ. ID. No. 4; SEQ. ID. No. 5; SEQ. ID. No.
6; SEQ. ID. No. 7; SEQ. ID. No. 8; SEQ. ID. No. 9; SEQ. ID. No. 10;
SEQ. ID. No. 11; SEQ. ID. No. 12; SEQ. ID. No. 13; SEQ. ID. No. 14;
SEQ. ID. No. 15; SEQ. ID. No. 16; SEQ. ID. No. 17; SEQ. ID. No. 18;
SEQ. ID. No. 19; SEQ. ID. No. 20; SEQ. ID. No. 21; SEQ. ID. No. 22;
SEQ. ID. No. 23; SEQ. ID. No. 24; SEQ. ID. No. 25; SEQ. ID. No. 26;
SEQ. ID. No. 27 and SEQ. ID. No. 28; but also the raw material from
which the said peptides can be obtained or are initially present in
the whole hydrolyzed products and fractions obtained from the
initial cocoa having bioactive properties, simultaneously showing
PEP enzyme inhibitory activity, and/or also antioxidant and/or
antineurodegenerative activity. Both the peptides and the extracts
containing them can be incorporated in functional foods as well as
forming part of pharmaceutical or dietetic compounds, and be used
to help in the treatment and prevention of different diseases,
especially cardiovascular disease and cerebral degeneration.
EXAMPLES
Example 1
[0036] Preparation of the cocoa extract, rich in bioactive
peptides. The Forastero cocoa variety from the Ivory Coast was used
to carry out the process.
[0037] The process of obtaining the bark was performed with fresh
cacao seeds obtained from cocoa pods by the process according to
the patent document ES 2286947 A1 and PCT/ES2008/000540, by the
same authors of the present invention. The product obtained after
defatting by pressing is referred to as bark if it is from beans
with the husk, as in the case of the present invention. To that
end, 100 g of bark, which had been defatted by pressing in a
continuous mechanical extractor, and had a residual fat content of
10%, was dissolved in 1 L of distilled water at a 1:10 (w:v) ratio
with a combination of Termamyl and Alcalase enzymes, at a
concentration of 1 .mu.l/g of each of them and maintained with
stirring for 1 hour at 50.degree. C. After this time had elapsed,
enzymes were inactivated at 100.degree. C. and centrifugation was
performed at 4000 rpm for 15 minutes and approximately 850 ml of
peptide-rich supernatant was recovered. This supernatant was used
to carry out bioactive peptide purification and PEP enzyme
inhibition assay.
Example 2
[0038] Purification of peptides from cocoa extract. The supernatant
containing the peptides and obtained after centrifugation was
subjected to two purification steps.
[0039] The first step of the process consisted of subjecting the
samples to a concentration phase by means of hydrophobic
interaction chromatography (HIC) in a AKTA Explorer chromatographer
(GE Healthcare, Amersham Biosciences AB). To that end the HiPrep
16/10 Phenyl FF (high sub) column was used and the balancing buffer
(100 mM sodium phosphate, 1.5 M (NH.sub.4).sub.2SO.sub.4, pH=7)
with a gradient of 1.5 at 0 M of (NH.sub.4).sub.2SO.sub.4 was used
for the subsequent elution. The said elution was monitored at 214
nm and PEP inhibitory activity was evaluated in vitro in each of
the 10 ml fractions obtained.
[0040] The fractions having activity were subjected to a
purification step by means of a first ultrafiltration step using 10
kDa filters (Amicon Ultra, Millipore), and subsequent reverse phase
chromatography (RPC) of the fraction below 10 kDa, using the
RESOURCE RPC 3 ml column (Amersham Biosciences) in an AKTA Explorer
chromatographer (Amersham Biosciences) and using a broad elution
gradient with eluents 0.1% TFA in miliQ water (A) and 0.1% TFA in
acetonitrile (B). The samples were monitored at 214 nm and, after
removing the solvents used in the process, PEP inhibitory activity
was re-evaluated in vitro in each of the fractions obtained, which
were of 2 ml in this case.
Example 3
Evaluation of PEP Enzyme Inhibitory Activity In Vitro
A.--Evaluation of PEP Inhibitory Activity.
[0041] Prolyl endopeptidase (PEP) enzyme activity was measured
according to the method described by Yoshimoto (Yoshimoto, T. and
Tsuru, D. 1978. Agr. Biol. Chem., 42, 2417; Yoshimoto, T., Walter,
R. and Tsuru, D. 1980. J. Biol. Chem., 255, 4786), based on the use
of substrate Z-Gly-Pro-p-nitroaniline, from which p-nitroaniline is
released due to the action of the PEP enzyme, which can be
spectrophotometrically quantified by taking absorbance readings at
410 nm.
[0042] PEP inhibitory activity was determined by adding the test
sample to the reaction mixture. The initial bark inhibition was
determined as control without enzymatic treatment, as well as after
protein hydrolysis with commercial enzymes and the purification HIC
fractions (FIG. 1).
[0043] Different fractions were separated by hydrophobic
interaction chromatography (HIC). Table 1 shows the PEP inhibition
percentage of the bark samples and of the different fractions.
[0044] Table 1 contains the results of PEP enzyme inhibition of all
the samples throughout the first step of purification.
TABLE-US-00002 TABLE 1 PEP inhibition percentage of bark samples as
well as the hydrophobic interaction fractions. Sample PEP
inhibition Untreated bark 22.81 .+-. 6.45 Alcalase + Termamyl bark
42.81 .+-. 2.98 Filtered bark (150 ml) 27.02 .+-. 1.49 Non retained
HIC 14.39 .+-. 1.49 F2 HIC 3.86 .+-. 1.47 F3 HIC 18.95 .+-. 8.93 F4
HIC 35.44 .+-. 17.37 F5 HIC 1.40 .+-. 0.98 F6 HIC 1.40 .+-. 0.91 F7
HIC 0.0 .+-. 0.0
Fractions 3 and 4, obtained subsequent to purification by
hydrophobic interaction chromatography, were selected as having the
greatest PEP enzyme inhibitory ability. Accordingly, they were
subjected to reverse phase chromatography (FIGS. 2 and 3). Tables 2
and 3 contain the PEP enzyme inhibition values of the fractions
obtained by reversed-phase chromatography from the hydrophobic
interaction fractions 3 and 4, respectively, as well as the protein
content of each.
TABLE-US-00003 TABLE 2 Percentage of PEP inhibition and protein
quantity of the RPC fractions obtained from hydrophobic interaction
fraction 3. Samples Protein content (mg) % PEP inhibition F6 RPC.
(F3 HIC) 0.041168081 10.18 .+-. 0.85 F7 RPC. (F3 HIC) 0.520117841
14.37 .+-. 3.39 F8 RPC. (F3 HIC) 5.329239984 24.55 .+-. 2.54 F9
RPC. (F3 HIC) 2.5620785 22.16 .+-. 0.85 F10 RPC. (F3 HIC)
1.131518808 19.16 .+-. 0.00 F11 RPC (F3 HIC) 0.470186129 8.38 .+-.
3.39 F12 RPC. (F3 HIC) 0.143528 10.78 .+-. 3.39 F13 RPC. (F3 HIC)
0.058311881 6.59 .+-. 7.62 F20 RPC. (F3 HIC) 0.039742304 8.38 .+-.
5.08
TABLE-US-00004 TABLE 3 Percentage of PEP inhibition and protein
quantity of the RPC fractions from hydrophobic interaction fraction
4. Sample Protein content (mg) % PEP inhibition F6 RPC. (F4 HIC)
0.013445155 11.65 .+-. 5.11 F7 RPC. (F4 HIC) 0.393839581 20.48 .+-.
0.57 F8 RPC. (F4 HIC) 6.269268373 34.54 .+-. 2.27 F9 RPC. (F4 HIC)
3.61016316 38.96 .+-. 1.70 F10 RPC. (F4 HIC) 1.97302672 26.51 .+-.
3.41 F11 RPC. (F4 HIC) 0.889492386 22.49 .+-. 1.14 F12 RPC. (F4
HIC) 0.299741375 19.28 .+-. 0.00 F13 RPC. (F4 HIC) 0.100727615
13.25 .+-. 3.98
Since RPC fractions 8, 9 and 10 from the HIC fraction 4 showed the
highest PEP enzyme inhibition values, they were selected for the
evaluation of peptide content therein. The said fractions were
analyzed by mass spectrometry MS/MS and 28 new peptides were
identified, whose sequences are listed in Table 4.
TABLE-US-00005 TABLE 4 Sequences of the 28 peptides identified in
RPC fractions 8, 9 and 10, from HIC fraction 4 (peptides 8 and 12
were identified in both fractions 9 and 10). MW (Da) Peptide
sequence Fraction 8 FI (F4HIC) SEQ.ID. No. 01 1067.37 GVKGEPGPNTL
SEQ.ID. No. 02 1173.45 ALPVNSPGKYE SEQ.ID. No. 03 1283.41
TDGVKGEPGPNTL SEQ.ID. No. 04 1292.45 LSQSPVYSNQNG SEQ.ID. No. 05
1483.69 VTTDGVKGEPGPNTL Fraction 9 FI (F4HIC) SEQ.ID. No. 06
1017.33 SDDDGQIRL SEQ.ID. No. 07 1053.31 NYDNSAGKW SEQ.ID. No. 08
1168.41 DNYDNSAGKW SEQ.ID. No. 09 1283.53 TDGVKGEPGPNTL SEQ.ID. No.
10 1437.47 RLDNYDNSAGKW SEQ.ID. No. 11 1483.67 VTTDGVKGEPGPNTL
SEQ.ID. No. 12 1857.83 ANSPVLDTDGDELQTGVQ Fraction 10 FI (F4HIC)
SEQ.ID. No. 13 777.27 GHAVTFF SEQ.ID. No. 14 904.31 FASKDQPL
SEQ.ID. No. 15 954.31 TFGEFQQV SEQ.ID. No. 16 968.43 VAPAGHAVTF
SEQ.ID. No. 17 1029.35 KAPLSPGDVF SEQ.ID. No. 18 1053.35 NYDNSAGKW
SEQ.ID. No. 08 1168.35 DNYDNSAGKW SEQ.ID. No. 19 1239.53
APLSPGDVFVAPA SEQ.ID. No. 20 1256.59 QVKAPLSPGDVF SEQ.ID. No. 21
1283.57 TDGVKGEPGPNTL SEQ.ID. No. 22 1367.73 KAPLSPGDVFVAPA SEQ.ID.
No. 23 1384.61 QQVKAPLSPGDVF SEQ.ID. No. 24 1594.93
QVKAPLSPGDVFVAPA SEQ.ID. No. 25 1629.81 SQSPVYSNQNGRFF SEQ.ID. No.
26 1669.41 WVTTDGVKGEPGPNTL SEQ.ID. No. 27 1758.79 SQSPVYSNQNGRFFE
SEQ.ID. No. 12 1857.83 ANSPVLDTDGDELQTGVQ SEQ.ID. No. 28 2020.93
ANSPVLDTDGDELQTGVQY
In Vivo Evaluation of RPC (Reverse Phase Chromatography)
Fractions
[0045] A. Evaluation of the Antineurodegenerative Capacity of the
Fractions in C.elegans.
[0046] The aim of this study was to evaluate in vivo the
functionality of the RPC fractions that gave positive results for
prolyl endopeptidase inhibition in vitro. With this purpose the
model organism Caenorhabditis elegans was used. The said
functionality is related to the potential protection provided by
the said extracts against developing Alzheimer's neurodegenerative
disease. To evaluate this capacity, RPC fractions 8, 9 and 10 from
HIC fraction 4 were selected and a test was performed to determine
body paralysis using C. elegans CL4176 transgenic strain. This
strain is characterised in that it expresses the human amyloid-beta
peptide (Ab1-42) after temperature induction.
[0047] Previously published data in the literature indicate that
the formation of amyloid beta-peptide plaques is preceded by
oxidative stress (Drake et al., "Oxidative stress precedes
fibrillar deposition of Alzheimer's disease amyloid beta-peptide
(1-42) in a transgenic Caenorhabditis elegans model" Neurobiol
Aging 2003, 24(3):415-20). Therefore it is interesting to evaluate
whether the effect exerted by the action of a molecule or compound
provides greater resistance to the accumulation in fibrillar A-beta
peptide deposits in the neurons. In these transgenic nematodes the
expression of the said peptide causes bodily paralysis that can be
evaluated after adding the molecule or compound of interest.
[0048] To conduct the experiment, age synchronized CL4176 strain
nematodes were obtained by plate cultivation on NGM (nematode
growth medium) at 16.degree. C. Eggs were collected on NGM plates
containing 100 .mu.L of each peptide fraction (F8, F9 and F10).
Positive controls were Z-prolyl prolinal (100 .mu.L of a 10 mM
stock on the plate surface) at a final concentration of 0.1 mM; and
vitamin C (25 .mu.L of a 0.04 mg/ml stock) at a final concentration
of 0.1 .mu.g/ml. Negative controls were NGM and NGM-non induced
(nematodes were incubated at 16.degree. C. throughout the
experiment). One hundred nematodes were analysed per condition.
After 1 day of incubation (when nematodes were approximately 23
hours old), temperature was increased from 16.degree. C. to
25.degree. C. to induce Ab peptide expression. Then 24 hours after
induction, temperature was dropped to 20.degree. C. and maintained
for the rest of the trial. After induction, the number of paralyzed
nematodes was analyzed for each condition tested until the
percentage of paralyzed nematodes reached 100%.
[0049] FIG. 4 shows the results obtained on completion of the body
paralysis trial using C. elegans CL4176 strain. It is evident that
the fractions studied (F8, F9 and F10) provide protection against
amyloid-beta peptide accumulation compared to the NGM-induced
control, with fractions F9 and F10 conferring the greatest
protection, at similar levels. The nematodes not induced with
amyloid-beta peptide do not suffer paralysis.
B. Evaluation of the Antioxidant Capacity of the Peptide Fractions
in C.elegans.
[0050] To conduct the experiment, age synchronized CL2070 strain
nematodes were obtained by plate cultivation on NGM at 20.degree.
C. Eggs were collected on NGM plates containing 100 .mu.L of each
peptide fraction (F8, F9 and F10). Positive controls were Z-prolyl
prolinal (100 .mu.L of a 10 mM stock on the plate surface) at a
final concentration of 0.1 mM; and vitamin C (25 .mu.L of a 0.04
mg/ml stock) at a final concentration of 0.1 .mu.g/ml. Negative
control was NGM. One hundred nematodes were assayed per condition.
After 7 days of incubation under the said conditions, the nematodes
were subjected to oxidative stress. To this end, nematodes were
transferred to plates with S basal medium containing H.sub.2O.sub.2
(2 mM). The plates were incubated at 20.degree. C. and after 5
hours the total number of nematodes surviving the treatment was
recorded.
[0051] FIG. 5 shows the increase in survival of nematodes treated
with peptide extracts F8, F9 and F10 after being subjected to
oxidative stress with H.sub.2O.sub.2 (2 mM) for 5 hours, as
compared to the negative NGM control.
C. Evaluation of C.elegans Survival after Addition of the Peptide
Fractions to the Culture Medium.
[0052] To conduct the experiment, age synchronized CL2070 strain
nematodes were obtained by plate cultivation on NGM at 20.degree.
C. Eggs were collected on NGM plates containing 100 .mu.L of each
peptide fraction (F8, F9 and F10). Positive controls were Z-prolyl
prolinal (100 .mu.L of a 10 mM stock on the plate surface) at a
final concentration of 0.1 mM; and vitamin C (25 .mu.L of a 0.04
mg/ml stock) at a final concentration of 0.1 .mu.g/ml. Negative
control was NGM. One hundred nematodes were assayed per condition.
Every 2 days the number of nematodes surviving in each condition
was counted until there were practically no living nematodes
left.
[0053] FIG. 6 shows the survival curve for nematodes grown in
different conditions for 21 days. It can be noted that both the
positive controls and the peptide fractions prolong the life of the
nematodes when compared with the negative NGM control.
Sequence CWU 1
1
36111PRTArtificial SequenceBiopeptido 1Gly Val Lys Gly Glu Pro Gly
Pro Asn Thr Leu1 5 10211PRTArtificial SequenceBiopeptido 2Ala Leu
Pro Val Asn Ser Pro Gly Lys Tyr Glu1 5 10313PRTArtificial
SequenceBiopeptido 3Thr Asp Gly Val Lys Gly Glu Pro Gly Pro Asn Thr
Leu1 5 10412PRTArtificial SequenceBiopeptidos 4Leu Ser Gln Ser Pro
Val Tyr Ser Asn Gln Asn Gly1 5 10515PRTArtificial
SequenceBiopeptidos 5Val Thr Thr Asp Gly Val Lys Gly Glu Pro Gly
Pro Asn Thr Leu1 5 10 1569PRTArtificial SequenceBiopeptidos 6Ser
Asp Asp Asp Gly Gln Ile Arg Leu1 579PRTArtificial
SequenceBiopeptidos 7Asn Tyr Asp Asn Ser Ala Gly Lys Trp1
5810PRTArtificial SequenceBiopeptidos 8Asp Asn Tyr Asp Asn Ser Ala
Gly Lys Trp1 5 10913PRTArtificial SequenceBiopeptidos 9Thr Asp Gly
Val Lys Gly Glu Pro Gly Pro Asn Thr Leu1 5 101012PRTArtificial
SequenceBiopeptido 10Arg Leu Asp Asn Tyr Asp Asn Ser Ala Gly Lys
Trp1 5 101115PRTArtificial SequenceBiopeptido 11Val Thr Thr Asp Gly
Val Lys Gly Glu Pro Gly Pro Asn Thr Leu1 5 10 151218PRTArtificial
SequenceBiopeptido 12Ala Asn Ser Pro Val Leu Asp Thr Asp Gly Asp
Glu Leu Gln Thr Gly1 5 10 15Val Gln137PRTArtificial
SequenceBiopeptido 13Gly His Ala Val Thr Phe Phe1 5148PRTArtificial
SequenceBiopeptido 14Phe Ala Ser Lys Asp Gln Pro Leu1
5158PRTArtificial SequenceBiopeptido 15Thr Phe Gly Glu Phe Gln Gln
Val1 51610PRTArtificial SequenceBiopeptido 16Val Ala Pro Ala Gly
His Ala Val Thr Phe1 5 101710PRTArtificial SequenceBiopeptido 17Lys
Ala Pro Leu Ser Pro Gly Asp Val Phe1 5 10189PRTArtificial
SequenceBiopeptido 18Asn Tyr Asp Asn Ser Ala Gly Lys Trp1
51913PRTArtificial SequenceBiopeptido 19Ala Pro Leu Ser Pro Gly Asp
Val Phe Val Ala Pro Ala1 5 102012PRTArtificial SequenceBiopeptido
20Gln Val Lys Ala Pro Leu Ser Pro Gly Asp Val Phe1 5
102113PRTArtificial SequenceBiopeptido 21Thr Asp Gly Val Lys Gly
Glu Pro Gly Pro Asn Thr Leu1 5 102214PRTArtificial
SequenceBiopeptido 22Lys Ala Pro Leu Ser Pro Gly Asp Val Phe Val
Ala Pro Ala1 5 102313PRTArtificial SequenceBiopeptido 23Gln Gln Val
Lys Ala Pro Leu Ser Pro Gly Asp Val Phe1 5 102416PRTArtificial
SequenceBiopeptido 24Gln Val Lys Ala Pro Leu Ser Pro Gly Asp Val
Phe Val Ala Pro Ala1 5 10 152514PRTArtificial SequenceBiopeptido
25Ser Gln Ser Pro Val Tyr Ser Asn Gln Asn Gly Arg Phe Phe1 5
102616PRTArtificial SequenceBiopeptido 26Trp Val Thr Thr Asp Gly
Val Lys Gly Glu Pro Gly Pro Asn Thr Leu1 5 10 152715PRTArtificial
SequenceBiopeptido 27Ser Gln Ser Pro Val Tyr Ser Asn Gln Asn Gly
Arg Phe Phe Glu1 5 10 152819PRTArtificial SequenceBiopeptido 28Ala
Asn Ser Pro Val Leu Asp Thr Asp Gly Asp Glu Leu Gln Thr Gly1 5 10
15Val Gln Tyr299PRTArtificial SequenceBiopeptido descrito en la
solicitud de patente PCT/ES2008/000540 29Ser Asp Asn Glu Trp Ala
Trp Met Phe1 53010PRTArtificial SequenceBiopeptido descrito en la
solicitud de patente PCT/ES2008/000540 30Leu Ser Asp Asn Glu Trp
Ala Trp Met Phe1 5 103110PRTArtificial SequenceBiopeptido descrito
en la solicitud de patente PCT/ES2008/000540 31Ser Asp Asn Glu Trp
Ala Trp Met Phe Lys1 5 103211PRTArtificial SequenceBiopeptido
descrito en la solicitud de patente PCT/ES2008/000540 32Leu Ser Asp
Asn Glu Trp Ala Trp Met Phe Lys1 5 103313PRTArtificial
SequenceBiopeptido descrito en la solicitud de patente
PCT/ES2008/000540 33Arg Arg Ser Asp Leu Asp Asn Gly Thr Pro Val Ile
Phe1 5 103413PRTArtificial SequenceBiopeptido descrito en la
solicitud de patente PCT/ES2008/000540 34Asp Asn Tyr Asp Asn Ser
Ala Gly Lys Trp Trp Val Thr1 5 103514PRTArtificial
SequenceBiopeptido descrito en la solicitud de patente
PCT/ES2008/000540 35Thr Ser Thr Val Trp Arg Leu Asp Asn Tyr Asp Asn
Ser Ala1 5 103615PRTArtificial SequenceBiopeptido descrito en la
solicitud de patente PCT/ES2008/000540 36Asp Asn Tyr Asp Asn Ser
Ala Gly Lys Trp Trp Val Thr Thr Asp1 5 10 15
* * * * *